The natural proteins are made up of naturally-occurring 20 amino acid species (hereafter referred to as “natural amino acids”). When protein structures or functions are analyzed or chemical behavior thereof is extended, amino acids that are not present in nature (hereafter referred to as “non-natural amino acids”) may be incorporated into desired positions of an amino acid sequence. Proteins into which non-natural amino acids have been incorporated are referred to as “alloproteins.”
Aminoacyl tRNA synthetase (hereafter referred to as “aaRS”) is an enzyme that binds a given amino acid specifically to given tRNA. Except for certain exceptional instances, 20 different types of such enzymes exist corresponding to each of 20 natural amino acid species. When alloproteins are to be synthesized, new aaRS corresponding to non-natural amino acids (hereafter referred to as “aaRS*”) and tRNA paired with a codon that does not encode natural amino acids (hereafter referred to as “tRNA*”) need to be incorporated into host cells to make them properly function therein. That is, tRNA* to which non-natural amino acids have been bound with the aid of aaRS* can be paired with a codon that does not naturally encode natural amino acids in host cells, in order to synthesize alloproteins into which non-natural amino acids have been incorporated.
In such a case, aaRS* is prepared based on existing aaRS that is specific for a given natural amino acid by modifying functions thereof so as to have activity of recognizing a non-natural amino acid similar to the given natural amino acid as a substrate. When aaRS* that is specific for O-methyltyrosine (i.e., a non-natural amino acid) similar to tyrosine (i.e., a natural amino acid) is to be prepared, for example, TyrRS mutant having enhanced specificity to o-methyltyrosine is prepared based on existing tyrosyl-tRNA synthetase (TyrRS). When alloproteins are synthesized with the use of such aaRS*, use of aaRS* that does not react with 20 natural amino acid species inherent in the host cells and tRNAs corresponding thereto but reacts specifically with given non-natural amino acid and tRNA* is necessary.
Thus, aaRS* having specificity to given non-natural amino acids, which is satisfactorily enhanced compared with specificity to existing natural amino acids, is used. This is because proteins into which natural amino acids have been introduced at sites into which given non-natural amino acids are to be introduced would be disadvantageously synthesized, otherwise. If aaRS* would react with tRNA that is inherent in the host cell besides tRNA*, non-natural amino acids would be introduced into sites into which natural amino acids should be introduced, besides sites into which non-natural amino acids are to be introduced. In order to avoid such problem, when prokaryotic cells are used as host cells, aaRS* that was constructed based on eukaryote-type aaRS may be used, because eukaryote-type aaRS is less likely to react with prokaryotic tRNA. The term “eukaryote-type aaRS” used herein refers to aaRS derived from eukaryotic organisms or aaRS derived from archaebacteria. If prokaryotic cells are used as host cells and prokaryote-derived aaRS* are introduced therein, such aaRS* may disadvantageously synthesize a plurality of types of aminoacyl tRNAs by recognizing tRNAs corresponding to natural amino acids inherent in the host cells as substrates, in addition to tRNA*. In such a case, unambiguous translation of a gene into a protein becomes difficult because of the aforementioned reasons. When prokaryotic host cells are used, accordingly, eukaryote-type aaRS* are to be used. When eukaryote-type cells are used as host cells, aaRS* prepared based on prokaryote-derived aaRS are used.
When alloproteins are synthesized, accordingly, adequate aaRS* needs to be prepared depending on whether the host cells to be used are eukaryotic or prokaryotic cells. aaRS* that can be used regardless of whether the host cells are eukaryote-type or prokaryotic cells rarely exists. When synthesis of alloproteins into which given non-natural amino acids have been incorporated is intended in eukaryote-type and prokaryotic cells, accordingly, preparation of prokaryote-derived aaRS* and eukaryote-type aaRS* is necessary. Preparation of aaRS*, however, requires modification of existing aaRS functions so as to realize activity of recognizing non-natural amino acids as substrates, which disadvantageously necessitates a large amount of labor.
Patent Document 1: WO 2003/014354
Patent Document 2: WO 2004/039989